Glycosaminoglycans (GAGs) which are part of the extra cellular matrix (ECM) can be chemically modified and adapted for medical use.
Hyaluronic acid (HA), which is the only known unsulfated GAG, is a ubiquitous component of the ECM of all connective tissues. It is a linear polysaccharide composed of a disaccharide repealing unit. The components of the disaccharide unit are N-acetyl-D-glucosamine and D-glucuronic acid linked by β1-4 and β1-3 linkages. HA has a range of naturally occurring molecular masses from several thousands to over 10 million Daltons.
Due to its unique physiochemical properties, HA has been implicated in water homeostasis of tissues, in regulating the permeability of other substances and in lubricating joints. HA binds specifically to proteins in the ECM and on the cell surface. These interactions are important in stabilizing the cartilage matrix, in cell motility, in cellular proliferation, in wound healing, in inflammation as well as in cancer metastasis (Morra, Biomacromolecules 6:1205-1223, 2005; Vercruysse and Prestwich, Critical Reviews in Therapeutic Drug Carrier Systems 15(5):513-555, 1998; Entwistle et al., J. Cell Biochem. 61:569-577, 1996). The unique viscoclastic nature of HA along with its biocompatibility and non-immunogenicity has led to its use in a number of clinical applications, which include: treatment of osteoarthritis of the knee, surgical aid in eye surgery, and healing and regeneration of surgical wounds (Goldberg and Buckwalter, Osteoarthritis Cartilage 13(3):216-224, 2005; Brown and Jones, J. Eur. Acad. Dermatol. Venereol. 19(3):308-318, 2005).
A variety of chemical modifications of native HA have been proposed to improve its mechanical and chemical properties. The principal targets for chemical modifications of HA are the hydroxyl and carboxyl functions.
Modifications via the hydroxyl function are mainly used for preparation of cross-linked HA by reaction with bifunctional cross-linkers e.g. divinyl sulfone and diglycidyl ethers (U.S. Pat. Nos. 4,582,865 and 4,713,448).
Modifications of the carboxylic functions are mainly used to introduce pendant functionalities that further permit attachment of drugs and biochemical reagents (Li et al., Biomacromolecules 5:895-902, 2004; Shu et al., J. Biomed. Mater. Res. 68A:365-375, 2004). Modifications of the carboxylic groups can also be used to obtain cross-linked products (Bulpitt and Aeschlimann, J. Biomed. Mater. Res. 47:152-169, 1999).
These modifications are made using hydrazides or amines. The activation of the carboxylic functions of HA towards nucleophilic attack by hydrazides or amines, in aqueous media, is mainly performed by the use of water soluble carbodiimides, especially 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC). Two major procedures for performing this activation are known in the art. The first one was developed by Prestwich et al. and is disclosed in U.S. Pat. No. 5,616,568, U.S. Pat. No. 5,874,417, (Prestwich et al., J. Controlled Release 53:93-103, 1998, and Pouyani and Prestwich, Bioconjugate Chem. 5:339-347, 1994). According to this procedure, HA is reacted with EDC under mildly acidic conditions (e.g. pH 4.75), to produce an active unstable O-acylisourea intermediate. Hydrazides that have a low pKa of 3-4 and retain their nucleophilicity at pH 4.75 efficiently react with the O-acylisourea intermediate to produce hydrazido derivatives of the glucuronic acid residues. In contrast, primary amines which are not nucleophiles at this pH failed to react with the active intermediate which eventually rearranges to a stable N-acylurea derivative.
The use of dihydrazide compounds such as adipic dihydrazide (ADH) provided derivatives of the formula HA-CO—NH—NH—CO—(CH2)4—CO—NH—NH2 (HA-ADH) having multiple pendant hydrazido groups for further derivatization with drugs, biochemical probes and cross-linking reagents.
Later publications demonstrated conjugation of the antitumor drug Taxol to the HA-ADH derivative (Luo and Prestwich, Bioconjugate Chem. 10:755-763, 1999) and the preparation of hydrogel films as bio-interactive dressings for wound healing from the HA-ADH derivative cross-linked with poly(ethyleneglycol)propiondialdehyde (Kirker et al., Biomaterials 23:3661-3671, 2002).
A second procedure for the activation of the carboxylic functions of HA was developed by Bulpitt and Aeschlimann (J. Biomed. Mater. Res. 47:152-169, 1999) and U.S. Pat. No. 6,630,457. According to this procedure, HA is reacted at pH 6.8 with a combination of EDC and the additive 1-hydroxybenzotriazole (HOBt). Initially, the carbodiimide and the carboxylate anion react to produce an active unstable O-acylisourea intermediate, which further reacts with the additive to form a more hydrolysis-resistant and non-rearrangeable active ester. This active ester readily reacts with hydrazides as well as with certain animes (which are present in an unprotonated form at pH of about 5.5-7.0). The use of this procedure allows the formation of HA derivatives with pendant hydrazido, amino as well as other functional groups.
The abovementioned methods for the introduction of pendant hydrazido groups with adipic dihydrazide (ADH) introduce a non-natural linker entity into the hyaluronic acid. The use of such derivatives for clinical applications inherently introduces these non-natural linker entities into the (human) organism which can lead to unexpected complications. It is therefore highly desirable to avoid the use of such linker moieties. The current invention presents a way to introduce hydrazido groups into HA while avoiding the use of linkers altogether, thereby circumventing possible complications indicated above.